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A Structural Insight into Major Groove Directed Binding
of Nitrosourea Derivative Nimustine with DNA: A
Spectroscopic Study
Shweta Agarwal1, Deepak Kumar Jangir1, Ranjana Mehrotra1*, Neelam Lohani2, M. R. Rajeswari2
1 Quantum Optics and Photon Physics, CSIR-National Physical Laboratory, New Delhi, India, 2 Department of Biochemistry, All India Institute of Medical Sciences, New
Delhi, India
Abstract
Nitrosourea therapeutics occupies a definite place in cancer therapy but its exact mechanism of action has yet to be
established. Nimustine, a chloroethyl nitrosourea derivative, is used to treat various types of malignancy including gliomas.
The present work focuses on the understanding of nimustine interaction with DNA to delineate its mechanism at molecular
level. Attenuated total reflection-Fourier transform infrared (ATR-FTIR) has been used to determine the binding sites of
nimustine on DNA. Circular dichroism (CD) spectroscopy has been used to confirm conformational variations in DNA
molecule upon nimustine-DNA interaction. Thermodynamic parameters of nimustine-DNA reaction have been calculated by
isothermal titration calorimetry. Results of the present study demonstrate that nimustine is not a simple alkylating agent
rather it causes major grove-directed-alkylation. Spectroscopic data suggest binding of nimustine with nitrogenous bases
guanine (C6 = O6) and thymine (C4 = O4) in DNA major groove. CD spectra of nimustine-DNA complexes point toward the
perturbation of native B-conformation of DNA and its partial transition into C-form. Thermodynamically, nimustine-DNA
interaction is an entropy driven endothermic reaction, which suggests hydrophobic interaction of nimustine in DNA-major
groove pocket. Spectral results suggest base binding and local conformational changes in DNA upon nimustine interaction.
Investigation of drug-DNA interaction is an essential part of rational drug designing that also provides information about
the drug’s action at molecular level. Results, demonstrated here, may contribute in the development of new nitrosourea
therapeutics with better efficacy and fewer side effects.
Citation: Agarwal S, Jangir DK, Mehrotra R, Lohani N, Rajeswari MR (2014) A Structural Insight into Major Groove Directed Binding of Nitrosourea Derivative
Nimustine with DNA: A Spectroscopic Study. PLoS ONE 9(8): e104115. doi:10.1371/journal.pone.0104115
Editor: Annalisa Pastore, National Institute for Medical Research, Medical Research Council, London, United Kingdom
Received April 14, 2014; Accepted July 5, 2014; Published August 7, 2014
Copyright: ß 2014 Agarwal et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper.
Funding: This work was supported by National Physical Laboratory, New Delhi. The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* Email: [email protected]
and methanol, representing its affinity to lipid bilayer membrane,
which enables it to cross the blood-brain-barrier for the
chemotherapy of gliomas [7]. Despite the availability of detailed
structural/chemical knowledge of the alkylating agents in the
literature, there remain deficits in the understanding of nimustineDNA interaction. Experimental evidence has indicated the
correlation of O6-methylguanine-DNA methyl transferase
(MGMT) gene expression level with cellular response to nimustine
[8]. Mineura et al carried out in vitro interaction studies between
nitrosoureas (involving nimustine) and Hind III digested cellular
DNA fragments. Subsequently, on piperidine hydrolysis, they
found that nimustine makes scission in DNA fragments corresponding to the location of guanine [9]. The formation of double
stranded breaks (DSB) in response to nimustine interaction with
DNA has already been reported [10]. Further, observations on
specific components activity of DSB-repair pathway suggest that
low activity of DNA ligase IV increases cell lethality towards
nimustine [10]. Despite the immense importance and direct
relevance of nimustine-DNA interactions, the underlying molecular mechanism of nimustine interaction with DNA has not been
explored so far.
Introduction
Most of the anticancer agents, currently used, have DNA and
auxiliary processes as their main target in the cell [1]. Therefore, a
comprehensive understanding on the physical/chemical interactions between DNA and small molecules (drug) becomes vital in an
effort to search potential drug candidates for targeted therapy [2].
Results of such investigations can suggest the modification of the
drug molecule in a way that produces fewer side effects and more
efficiency [3]. Interaction studies can improve the understanding
on the binding mechanism of the drug with its target molecule.
Further, such investigations can offer details on the moieties that
are involved in the interaction [3]. Alkylating agents comprise a
major class of therapeutics, which are used in the treatment of
different types of cancer [4]. Nimustine or ACNU [(1-(4-amino-2methyl-5-pyrimidynyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride)] (Figure 1), one of the derivatives of nitrosourea, is
used as an alkylating anticancer drug [5]. It is a cell-cycle phase
nonspecific antineoplastic agent, mainly used for the treatment of
malignant gliomas (brain/spine tumor) [5]. Nimustine, in addition
to radiation therapy, provides a major therapeutic option for the
high-grade gliomas [6]. Nimustine is soluble both in both water
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Binding of Nitrosourea Derivative Nimustine with DNA
Materials and Methods
Sample Preparation
Nimustine (M.W- 272.69) and highly polymerized type I calf
thymus DNA were procured from Sigma Aldrich chemicals, USA.
Ratio of the absorbance of DNA at 260 nm (A260) and 280 nm
(A280) was used to determine the purity of DNA. The calculated
ratio of (A260)/(A280) in DNA sample was found to be 1.81,
suggesting the sufficient purity of DNA [21]. Other reagents and
chemicals utilized in this investigation were of analytical grade.
Deionized ultra pure water (Scholar-UV Nex UP 1000 system)
having resistance of 18.2 M was used for the preparation of buffer
solution and nimustine drug solutions. Stock solution of DNA
sodium salt was prepared in 10 mM tris-HCl buffer (pH 7.4). This
solution was placed at 8uC for 24 hour in conjunction with stirring
at regular intervals for maintaining the homogeneity of DNA
solution. Final concentration of DNA stock solution was measured
spectrophotometrically using molar extinction coefficient of
6600 cm21 M21 [22]. The final concentration of DNA stock
solution was 42 mM due to molarity of phosphate group.
ATR-FTIR Spectroscopic Measurement
For studying nimustine-DNA interaction, nimustine solution of
varying concentration was added separately dropwise into DNA
solution of constant concentration (42 mM) to attain 1/60, 1/40
and 1/20 molar ratios (r). This is followed by continous vortexing
for 15 minutes and incubation at room temperature for two hour
to ensure the complexation of nimustine with DNA. FTIR spectral
measurements of free calf thymus DNA and nimustine-DNA
complexes were recorded on Varian-660-IR spectrophotometer
equipped with KBr beam splitter and deuterated triglycine
sulphate (DTGS) detector. Continuous purging of dry nitrogen
gas was performed to remove water vapors from sample chamber.
For the sampling in ATR mode, Miracle (PIKE) ZnSe-micro
horizontal attenuated total internal reflection (HATR) assembly
was used. Ambient humidity of 46% RH was maintained during
the experiments. Two hundred fifty six interferograms with a
resolution of 2 cm21 were collected in the spectral range of 2400–
700 cm21. Before the recording of each measurement, background atmospheric spectrum was collected. No data treatment
was performed except multiple baseline correction, water
subtraction and normalization for DNA band at 968 cm21. To
execute water subtraction, a spectrum of tris buffer was recorded
and then subtracted from the spectra of free DNA and nimustineDNA complexes. An acceptable water subtraction was achieved
when the intensity of water combination band at about 2200 cm21
became zero in all the spectra collected [23]. Infrared spectrum of
free nimustine was also recorded (Figure 2) and subtracted from
the nimustine-DNA complexes spectra. This was done to make
sure that observed spectral variations in DNA are due to nimustine
binding.
Figure 1. Chemical structure of nimustine.
doi:10.1371/journal.pone.0104115.g001
Significant developments have been made over the past few
years in the area of drug-DNA interactions using different
biophysical methods and spectroscopic techniques [11,12]. Attenuated total reflection-Fourier transform infrared (ATR-FTIR)
spectroscopy has emerged as an efficient tool in providing the
whole information on biomolecule structure and their complexes.
It is a fast technique, which provides results in a single snapshot
[13]. ATR-FTIR can be applied on short oligonucleotide to full
length DNA and can be used for liquid samples at physiological
mimic conditions. Recent advances in ATR-FTIR spectroscopy
have enabled its extensive use in investigation of nucleic acid
binding properties of small ligands and drugs [13–16]. In addition,
circular dichroism (CD) spectroscopy is highly sensitive technique
to determine conformational transition in biomolecules. It can
even distinguish sub-conformational isomerization between distinct conformers [17,18]. Another parameter of paramount
importance is thermodynamic profiling (enthalpy, entropy and
Gibb’s free energy) of drug-DNA complexes [1,19]. Isothermal
titration calorimetry (ITC) can reveal information on the nature of
forces that drive the complex formation. ITC is particularly
important for determining the binding affinity, enthalpy, entropy
and reaction stoichiometry with high accuracy [19,20].
In the present study, we have utilized the potential of ATRFTIR, CD spectroscopic techniques and ITC to study intimate
binding properties of nimustine to DNA. The structural evidence,
offered here, delivers essential information on the DNA interaction
properties of nimustine. This knowledge may provide inputs for
the formulation of new nitrosourea derivatives with anti-cancer
potential.
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CD Spectroscopic Measurement
CD spectral measurements were carried out on Applied
Photophysics (Chirascan) spectrophotometer. Spectra were collected in the far UV range (200 nm–320 nm) using quartz cuvette
having pathlength of 1 mm. Spectral collection was done at room
temperature after two hours incubation of nimustine with DNA.
For each sample, five scans were recorded with a scanning speed
of 1 nm/sec and then averaged. To perform subtraction, spectrum
of buffer was subtracted from the spectra of free DNA and
nimustine-DNA complexes. CD investigations were performed
using nimustine solution of various concentration in the range of
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Binding of Nitrosourea Derivative Nimustine with DNA
Figure 2. FTIR spectrum of free nimustine in the region of 1800 cm21 to 700 cm21.
doi:10.1371/journal.pone.0104115.g002
thymine (from 1657 cm21 to 1660 cm21). Band at 1493 cm21
(cytosine) shows 1 cm21 up-shift upon nimustine interaction with
DNA. Shifts in band position for the nitrogenous bases of DNA are
also found accompanied by the changes in intensity at all molar
ratios of nimustine-DNA complexes. Positive bands at 1724 cm21
and 1652 cm21 are observed in the difference spectra of
nimustine-DNA complexes [(DNA solution+nimustine solution)–
DNA solution] (Figure 4). These bands specify infrared hyperchroism for the stretching vibrations of guanine and thymine
respectively [13–15]. Minor increase in intensity of cytosine is also
evident by positive infrared features at around 1493 cm21. No
appreciable change in intensity and position of adenine band at
1609 cm21 is noticed in nimustine-DNA complexes. Deviations in
intensity and shift in the infrared bands associated with guanine
and thymine suggest direct interaction of nimustine with the
moieties of these heterocyclic nitrogenous bases [13–15]. Furthermore, groups C6 = O6 (guanine) and C4 = O4 (thymine) are
located in major groove of DNA. Therefore, spectral variations at
1715 cm21 and 1657 cm21 augment the possibility of nimustine
mechanism via ‘major groove-directed-alkylation’ [25]. The
plausible explanation of these spectral observations can be that
nimustine is first positioned within DNA major groove and then
performs alkylation via transfer of chloroethyl moiety (from
nimustine) to O6 position of guanine (Figure 5). Results are in
corroboration with the fact that ‘‘most of the alkylating agents are
major groove binder’’ [26]. Phenomenon of groove binding
followed by alkylation has also been observed in the case of
altromycin B [27] and anthramycin [28]. Along with the spectral
changes (shifts and intensity change), percent effect of nimustine
binding on four bands representative of reactive sites guanine
C6 = O6, thymine C4 = O4 (located in major groove), cytosine
and adenine is shown in Figure 6.
0.041–0.125 mM with constant DNA concentration of 2.5 mM to
attain 1/60, 1/40 and 1/20 molar ratios (r).
ITC Measurement
ITC was performed using NANO-ITC (isothermal titration
calorimeter-USA) system at 25uC temperature. Total twenty serial
injections of nimustine (1.5 mM) were added at the interval of
200 seconds to calf thymus DNA (561023 mM). A control
experiment was carried out to calculate the heat of dilution for
DNA into buffer (pH-7.4). The net enthalpy-entropy changes for
nimustine-DNA interaction was determined by subtracting the
corresponding heat of dilution derived from the injection of same
amount of nimustine into buffer alone.
Results and Discussion
FTIR Spectral Outcome
DNA Base Binding. The infrared spectral features observed
in the spectrum of free calf thymus DNA and nimustine-DNA
complexes are shown in Figure 3. Stretching vibrations due to
deoxyribose sugar, phosphate (PO2- symmetric and asymmetric)
and nitrogenous bases (C = O, C = N) of DNA lie in the region of
1800–700 cm21. The infrared band at 1715 cm21 is assigned to
guanine (G) due to in-plane stretching vibrations of C6 = O6
bonds [13–15,24]. The band at 1657 cm21 is attributed primarily
to thymine (T) stretching vibrations of C4 = O4 bonds [13–15,24].
The bands at 1609 cm21 and 1493 cm21 appear due to the ring
stretching vibrations of adenine (C = N) and cytosine (C = C)
respectively [13–15,24].
Infrared band observed in the spectrum of free DNA at
1715 cm21 (guanine) shows downshift of 3 cm21 (from
1715 cm21 to 1712 cm21) in nimustine-DNA complexes. Similarly, up-shift of 3 cm21 is observed in infrared band assigned to
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Binding of Nitrosourea Derivative Nimustine with DNA
Figure 3. FTIR spectra of free DNA and nimustine-DNA complexes. FTIR spectra of free calf thymus DNA and its complexes with nitrosourea
derivative nimustine at different molar ratios were collected in the region of 1800 cm21 to 700 cm21.
doi:10.1371/journal.pone.0104115.g003
Figure 4. Difference spectra of nimustine-DNA complexes in the region of 1800 cm21 to 700 cm21. {Difference spectra = [(DNA solution+
nimustine solution)–(DNA solution)]}.
doi:10.1371/journal.pone.0104115.g004
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Figure 5. The proposed model for binding of nimustine to DNA.
doi:10.1371/journal.pone.0104115.g005
1220 cm21 are observed, which indicate change in intensity of
phosphate stretching vibrations upon drug interaction. Deoxyribose sugar vibrations due to C = O and C-C stretching are
denoted by infrared bands at 1052 cm21 and 968 cm21 in the
spectrum of free calf thymus DNA [13–15,24]. No significant shift
is observed in these bands in the complexes however; both of these
bands show minor infrared hyperchroism (positive bands at
1043 cm21 and 956 cm21 in difference spectra). All of these
observations suggest slight external binding of nimustine with
In the spectrum of
free calf thymus DNA (Figure 3), infrared bands appeared at
1221 cm21 and 1084 cm21 are due to phosphate asymmetric and
symmetric stretching vibrations respectively [13–15,24]. Downward shift of 3 cm21 is observed in phosphate asymmetric
stretching vibrations band at 1221 cm21 at highest molar ratio
(1/20). No appreciable shift is noticed in the band of phosphate
symmetric vibrations at 1084 cm21. In the difference spectra
(Figure 4), positive band at 1086 cm21 and negative band at
Phosphate-Sugar Backbone Binding.
Figure 6. Percentage effect of nimustine on DNA major groove. Percentage effect of nimustine on DNA nitrogenous base guanine (GC6 = O6) 1715 cm21 and thymine (T-C4 = O4) 1657 cm21) and other bases of DNA adenine (A) and cytosine (C) was observed as a function of
nimustine concentration.
doi:10.1371/journal.pone.0104115.g006
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Therefore, altogether, the presence of these spectral features
augment a possibility of the perturbation of DNA conformation
from B (10.4 base pair/helical turn) to C-form (,9.4 base pair/
helical turn) [30,35,38]. However, it seems that the perturbation in
DNA conformation is limited to few base pairs. When complete B
to C transition occurs, then the CD band at 243 nm shows about
66% decrease in its intensity. However, not much decrease in the
concerned band is observed. Hence, there are possibilities of the
formation of an intermediate form of DNA having features of both
B and C conformation. Similar results have been observed in the
case of cationic lipid [30] and neutral lipid binding with DNA [39]
that have been ascribed to a non-cooperative augment in DNA
winding angle due to change in base pair per turn from 10.4 to
9.8. Moreover, increase in winding angle (or decrease in propeller
twist) causes widening in DNA groove [40] that enables proper
positioning of small ligands in the groove pocket. This suggests that
nimustine is partially positioned within the major groove and
modulates accessibility for alkylation of nitrogenous base via
transfer of chloroethyl moiety from drug to O6 position of
guanine. This is in accordance with FTIR results that signify the
interaction of nimustine with guanine (C6 = O6) and thymine
(C4 = O4), representative of major groove [41]. Bands attributed
to b-N-glycosidic linkage (at 214 nm) and hydrogen bonding
(224 nm) show no appreciable change in the complex spectra.
phosphate-sugar backbone of DNA double helix [13–15,24].
Bands at 1374 cm21, 1296 cm21, 779 cm21 and 724 cm21 are
assigned to sugar conformations [13–15,24] and show negligible
shift when the nimustine-DNA interaction takes place. In the
difference spectra of nimustine-DNA complexes, positive features
are observed at about 1347 cm21, 770 cm21 and 730 cm21 due
to the increase in intensity of sugar vibrations. Spectral changes
observed for the sugar conformations, deoxy ribose-phosphodiester chain vibrations and sugar-phosphate backbone stretching
vibrations suggest fine external binding of nimustine with DNA
[13–15].
DNA Conformation. Infrared band, emerges due to S-C2
endo/anti sugar pucker-phosphodiester stretching vibrations at
838 cm21, is regarded to be marker of B-conformation of DNA
(Figure 3) [24,29]. Moreover, infrared bands at 1221 cm21
(antisymmetric PO2 stretching) and 894 cm21 (deoxyribose ring
stretching) are also characteristic feature of B-DNA [24,29]. In
addition, conformation of C-N glycosidic linkage (infrared band at
1458 cm21) is also responsible for the maintenance of DNA in Bform [24,29]. Upon nimustine complexation with DNA, band at
1221 cm21 is shifted to lower frequency (1218 cm21), which
suggests a decrease in DNA hydration state [30]. The infrared
band at 1458 cm21 (glycosidic bond) is shifted to 1462 cm21 after
nimustine-DNA interaction. Besides this, there is an emergence of
new band at 872 cm21, which is considered a characteristic
feature of DNA in C-conformation [30]. Reduction in hydration
state around phosphate group of DNA with the appearance of new
band at 872 cm21 indicates the transition of duplex from B to Cform [30]. Nevertheless, this transition occurs at local level, as
evident by the presence of other prominent DNA B-form markers
(1220 cm21 and 837 cm21) in nimustine-DNA spectra. These
changes confirm that DNA remains globally in B-form.
Thermodynamic Profile of Nimustine-DNA Interaction
Figure 8 (a) shows the ITC titrations results, where each heat
burst curve represents to a single nimustine injection to calf
thymus DNA. The area below these curves is determined by
integration to yield the associated injection heats (net injection
heat). Net heat is then plotted against injection number depicted in
the lower panel (Figure 8b). The dots reflect the experimental
injection heat while the solid line represents the calculated fit of the
data set. The data have been fitted to the single set of identical sites
model that give a reasonable fitting of the experimental data. The
interaction data represents entropy driven endothermic binding
event with positive entropy change (DS) 2658 kJ/mol and positive
enthalpy change (DH) 764.260.12 kJ/mol. The determined
binding affinity constant (Ka) is found to be 9.864.56103/mole
with Gibb’s free energy (DG) 2773.3 kJ.
Nimustine-DNA interaction is favored by positive enthalpy
changes that indicate groove binding of nimustine into double
helix [42]. Furthermore, positive entropy suggests the disruption of
the unique water molecules lining the DNA groove [19].
Reasonable explanation of this outcome may be the hydrophobic
interaction of nimustine into major groove where it makes a stable
contact with DNA to perform alkylation [19,20]. This is in
complete agreement with spectroscopic results.
CD Spectral Outcome
Affirmation of DNA Conformational changes. Figure 7
shows the CD spectra of free calf thymus DNA and its complexes
with nimustine. Asymmetrical glycosidic bond and specific righthanded helical arrangement of B-DNA give a typical CD
spectrum that comprises two positive and two negative ellipticitical
components: 268 nm (positive), 243 nm (negative), 224 nm
(positive) and 214 nm (negative). Band at 268 nm arises due to
the stacking interaction between the nitrogenous bases while the
band at 243 nm is attributed to the right-handedness of B-DNA
[31–33]. Manifestation of bands at 214 nm and 224 nm is due to
b-N-glycosidic linkage (present between nitrogenous base and
deoxyribose sugar) and hydrogen bonds occurring between the
nitrogenous bases of opposite strands respectively [31–33].
Alteration in band position as well as in intensity of these spectral
bands is due to the corresponding conformational transitions in
duplex DNA allied to its interaction with drug. Upon the addition
of nimustine, the band at 268 nm (assigned to base stacking) shows
bathochromic shift (red shift) of 7 nm along with decrease in
positive ellipticity (32%). Furthermore, negative band (243 nm)
attributed to helicity shows red shift of 4 nm with reduction in
ellipticity (44%) at all molar ratios. Red shift and decrease in molar
ellipticity at these bands (268 nm and 243 nm) suggest the
distortion in native conformation of B-DNA due to nimustine
interaction [33]. A loss of 268 nm CD intensity along with red
shift has been correlated with small change in number of base pair
per turn in DNA helix [30,34–36] and reflects the increase in
DNA winding angle [37]. These spectral variations show the
presence of some C DNA features in native conformation of DNA
upon nimustine interaction. Moreover, reduction in 243 nm CD
band is considered a key marker of C-form of DNA [30,34–36].
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Conclusions
The spectroscopic results show that nimustine is a major groove
directed alkylating agent. Further analysis illustrates that nimustine
interaction occurs via guanine (C6 = O6) and thymine (C4 = O4)
reactive sites located in DNA major groove. Some degree of
external interaction with phosphate-sugar backbone has also been
observed. CD spectral results suggest the formation of an
intermediate form of DNA during the transition from B to Cform at local level after nimustine-DNA complex formation,
although globally DNA remains in native B-form. Thermodynamically nimustine-DNA interaction is found to be entropy
driven endothermic reaction. These findings may add to an
understanding about the interaction mode of nimustine with DNA
at molecular level.
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Binding of Nitrosourea Derivative Nimustine with DNA
Figure 7. Circular dichroism spectra of free DNA and nimustine -DNA complexes at different molar ratios.
doi:10.1371/journal.pone.0104115.g007
Figure 8. ITC curves for the binding of nimustine to DNA.
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Binding of Nitrosourea Derivative Nimustine with DNA
Acknowledgments
Author Contributions
The authors (SA, DKJ and RM) thank Director, CSIR-National Physical
Laboratory, New Delhi for granting the permission for publication of the
work.
Conceived and designed the experiments: SA DKJ RM NL MRR.
Performed the experiments: SA NL. Analyzed the data: SA DKJ RM.
Contributed reagents/materials/analysis tools: RM MRR. Contributed to
the writing of the manuscript: SA DKJ RM.
References
22. Vijayalakshmi R, Kanthimathi M, Subramanian V, Nair BU (2000) DNA
cleavage by a chromium (III) complex. Biochemical and biophysical research
communications 271: 731–734.
23. Alex S, Dupuis P (1989) FT-IR and Raman investigation of cadmium binding by
DNA. Inorganica Chimica Acta 157: 271–281.
24. Banyay M, Sarkar M, Gräslund A (2003) A library of IR bands of nucleic acids
in solution. Biophysical chemistry 104: 477–488.
25. Zewail-Foote M, Hurley LH (1999) Ecteinascidin 743: a minor groove alkylator
that bends DNA toward the major groove. Journal of medicinal chemistry 42:
2493–2497.
26. Pelengaris S, Khan M (2013) The Molecular Biology of Cancer: A Bridge from
Bench to Bedside: Wiley.com.
27. Hansen M, Hurley L (1995) Altromycin B Threads the DNA Helix Interacting
with Both the Major and the Minor Grooves to Position Itself for Site-directed
Alkylation and Guanine N7. Journal of the American Chemical Society 117:
2421–2429.
28. Pommier Y, Kohlhagen G, Bailly C, Waring M, Mazumder A, et al. (1996)
DNA sequence-and structure-selective alkylation of guanine N2 in the DNA
minor groove by ecteinascidin 743, a potent antitumor compound from the
Caribbean tunicate Ecteinascidia turbinata. Biochemistry 35: 13303–13309.
29. Ouameur AA, Tajmir-Riahi HA (2004) Structural analysis of DNA interactions
with biogenic polyamines and cobalt (III) hexamine studied by Fourier transform
infrared and capillary electrophoresis. Journal of Biological Chemistry 279:
42041–42054.
30. Braun CS, Jas GS, Choosakoonkriang S, Koe GS, Smith JG, et al. (2003) The
structure of DNA within cationic lipid/DNA complexes. Biophysical journal 84:
1114–1123.
31. Curtis-Johnson W, Nakanishi K, Berova N, Woody R (1994) CD of Nucleic
Acids in Circular Dichroism. Principles and Applications, VHS, New York:
523–540.
32. Miyahara T, Nakatsuji H, Sugiyama H (2012) Helical structure and circular
dichroism spectra of DNA: A theoretical study. The Journal of Physical
Chemistry A 117: 42–55.
33. Zhao C, Ren J, Gregoliński J, Lisowski J, Qu X (2012) Contrasting
enantioselective DNA preference: chiral helical macrocyclic lanthanide complex
binding to DNA. Nucleic acids research 40: 8186–8196.
34. Bokma JT, Curtis WJ, Blok J (1987) CD of the li-salt of DNA in ethanol/water
mixtures: Evidence for the B-to C-form transition in solution. Biopolymers 26:
893–909.
35. Portugal J, Subirana J (1985) Counterions which favour the C form of DNA.
The EMBO journal 4: 2403.
36. Zhang Z, Huang W, Tang J, Wang E, Dong S (2002) Conformational transition
of DNA induced by cationic lipid vesicle in acidic solution: spectroscopy
investigation. Biophysical chemistry 97: 7–16.
37. Chan A, Kilkuskie R, Hanlon S (1979) Correlations between the duplex winding
angle and the circular dichroism spectrum of calf thymus DNA. Biochemistry
18: 84–91.
38. Patil SD, Rhodes DG (2000) Conformation of oligodeoxynucleotides associated
with anionic liposomes. Nucleic acids research 28: 4125–4129.
39. Zuidam NJ, Barenholz Y, Minsky A (1999) Chiral DNA packaging in DNAcationic liposome assemblies. FEBS letters 457: 419–422.
40. Pullman B (1985) Specificity in the interaction of non intercalative groove
binding ligands with nucleic acids. Journal of biosciences 8: 681–688.
41. Xiong Y, Sundaralingam M (2001) Protein–nucleic acid interaction: major
groove recognition determinants. eLS.
42. Haq I (2002) Thermodynamics of drug-DNA interactions. Archives of
biochemistry and biophysics 403: 1–15.
1. Haq I, Ladbury J (2000) Drug–DNA recognition: energetics and implications for
design. Journal of Molecular Recognition 13: 188–197.
2. Kennard O (1993) DNA-drug interactions. Pure and applied chemistry 65:
1213–1222.
3. Chaires JB (1998) Drug–DNA interactions. Current opinion in structural biology
8: 314–320.
4. Puyo S, Montaudon D, Pourquier P (2013) From old alkylating agents to new
minor groove binders. Critical reviews in oncology/hematology.
5. Takakura K, Abe H, Tanaka R, Kitamura K, Miwa T, et al. (1986) Effects of
ACNU and radiotherapy on malignant glioma. Journal of neurosurgery 64: 53–
57.
6. Tanaka M, Shibui S, Nomura K, Nakanishi Y (2001) Radiotherapy combined
with nimustine hydrochloride and etoposide for malignant gliomas: results of a
pilot study. Japanese Journal of Clinical Oncology 31: 246–250.
7. Miyagami M, Tsubokawa T, Tazoe M, Kagawa Y (1990) Intra-arterial ACNU
chemotherapy employing 20% mannitol osmotic blood-brain barrier disruption
for malignant brain tumors. Neurologia medico-chirurgica 30: 582.
8. Tanaka S, Kobayashi I, Utsuki S, Oka H, Fujii K, et al. (2003) O6methylguanine-DNA methyltranspherase gene expression in gliomas by means
of real-time quantitative RT-PCR and clinical response to nitrosoureas.
International journal of cancer 103: 67–72.
9. Mineura K, Fushimi S, Itoh Y, Kowada M (1988) DNA lability induced by
nimustine and ramustine in rat glioma cells. Journal of Neurology, Neurosurgery
& Psychiatry 51: 1391–1394.
10. Kondo N, Takahashi A, Mori E, Noda T, Su X, et al. (2010) DNA ligase IV is a
potential molecular target in ACNU sensitivity. Cancer science 101: 1881–1885.
11. Chaires JB (1997) Energetics of drug–DNA interactions. Biopolymers 44: 201–
215.
12. Zerbe O (2012) First Solution Structures of Seven-Transmembrane Helical
Proteins. Angewandte Chemie International Edition 51: 860–861.
13. Jangir DK, Kundu S, Mehrotra R (2013) Role of Minor Groove Width and
Hydration Pattern on Amsacrine Interaction with DNA. PloS one 8: e69933.
14. Tyagi G, Pradhan S, Srivastava T, Mehrotra R (2014) Nucleic acid binding
properties of allicin: Spectroscopic analysis and estimation of anti-tumor
potential. Biochimica et Biophysica Acta (BBA) - General Subjects 1840: 350–
356.
15. Agarwal S, Jangir DK, Singh P, Mehrotra R (2013) Spectroscopic analysis of the
interaction of lomustine with Calf thymus DNA. Journal of Photochemistry and
Photobiology B: Biology.
16. Agarwal S, Jangir DK, Mehrotra R (2012) Spectroscopic studies of the effects of
anticancer drug mitoxantrone interaction with calf-thymus DNA. Journal of
Photochemistry and Photobiology B: Biology.
17. Kypr J, Kejnovská I, Renčiuk D, Vorlı́čková M (2009) Circular dichroism and
conformational polymorphism of DNA. Nucleic acids research 37: 1713–1725.
18. Vorlı́čková M, Kejnovská I, Bednářová K, Renčiuk D, Kypr J (2012) Circular
Dichroism Spectroscopy of DNA: From Duplexes to Quadruplexes. Chirality
24: 691–698.
19. Barceló F, Capó D, Portugal J (2002) Thermodynamic characterization of the
multivalent binding of chartreusin to DNA. Nucleic acids research 30: 4567–
4573.
20. Pagano B, Mattia CA, Giancola C (2009) Applications of isothermal titration
calorimetry in biophysical studies of G-quadruplexes. International journal of
molecular sciences 10: 2935–2957.
21. Glasel J (1995) Validity of nucleic acid purities monitored by 260 nm/280 nm
absorbance ratios. Biotechniques 18: 62–63.
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